This invention relates to a method for determining in a test sample one or more components of the reaction between a specifically-binding protein and the corresponding bindable substance, using the mutual reactivity of such components and of at least one labelled component obtained by coupling or adsorbing particles of a sol of the label directly to the component, comprising during, or after the completion of, the reaction, optionally after the separation of the bound and the free labelled component, determining in said test sample, or in one of the fractions obtained after separation, the presence and/or the quantity of the label by a method suitable for the purpose to obtain a qualitative or quantitative indication of the component to be determined.
The invention further relates to a method of preparing a labelled component of the reaction between a specific binding protein and the corresponding bindable substance by coupling or adsorbing particles of a sol of the label directly to the component, and to a test kit for use in the determination of an immuno component in an aqueous test sample.
As used herein, the phrase "component of the reaction between a specific binding protein and the corresponding bindable substance" means substances, or parts thereof, such as receptor proteins and antigenic determinants, which may be present at the surface of cells, and immuno chemical substances, such as haptens, antigens, and antibodies, which may be present in various media, in particular body fluids, such as blood plasma, serum, and the like or culturing media of cells.
The invention is accordingly concerned with a plurality of fields of histology, such as tissue and cell staining, in which immunochemical reactions take place, but also couplings or adsorptions may take place between the colloidal label and other (macro)molecular structures, such as enzymes, streptavidin, DNA and/or RNA, etc. In addition to these fields, the invention is also concerned with the field of immunoassays for, e.g., diagnostic purposes (determination of antibodies, antigens or haptens in aqueous test samples).
In the part of the specification which follows, the invention will be described in more detail with particular reference to the application of the invention to the field last mentioned, i.e., diagnostic immunoassays, but the invention should not be construed as being limited to such application, as it is equally applicable to histological and histochemical examination methods.
In EP-A-0007654, a survey is given of known immunochemical methods in which the presence of a given immunological component is determined qualitatively and/or quantitatively, using the mutual reactions between such components, such as the reaction between antigen and the antibody against it. These known methods each have certain disadvantages or drawbacks which, according to the above European patent application, can be removed by using, in a method as described above in the opening paragraph, a metal-labelled component, obtained by coupling or adsorbing the component directly or indirectly to particles of an aqueous dispersion of a metal or metal compound or of polymer nuclei coated with a metal or metal compound, with the particles having a size of 6-100 nm.
The metal-immunochemical technique described is more sensitive than the known radio- and enzyme-immunotechniques, and in addition renders it possible to demonstrate and determine more than one immunological component in the same test medium simultaneously by using different metal labels.
The metal sols may be of metals, or metal compounds, such as metal oxides, metal hydroxides or metal salts. As examples are mentioned the metals or metal compounds of gold, silver, iron, nickel, aluminum, chromium, lead, vanadium, mercury, manganese, and generally all those metals which can be readily demonstrated by means of known techniques. Sols of metals are, for example, those of silver, gold and platinum. Sols of metal compounds are, for example, those of silver iodide, iron oxide, aluminum hydroxide, chromium hydroxide, vanadium oxide, iron hydroxide, manganese hydroxide and mercury sulfide.
Preferably, metals or metal compounds are used which do not occur in the test medium, and of these specifically those which can be demonstrated with a selected technique in as low a concentration as possible.
In EP-A-0032270, a survey is given of the possibilities of a qualitative and/or quantitative determination of an immuno-chemical component, in which one or more labelled components are used which have been obtained by directly or indirectly coupling such a component or components to particles of an aqueous dispersion of a hydrophobic dye or pigment, or of polymeric nuclei coated with such a dye or pigment.
U.S. Pat. No. 4,760,030 (Peterson et al.) discloses a method for determining the presence of a specific binding pair member (sbp member) in a sample. The method involves an agglutination assay using opaque particles capable of agglutinating in the presence of the sbp member. The opaque particles may be derived from carbon particles having a particle size of from 0.2 to 5.0 microns. The carbon particles are conjugated to a specific binding partner of the sbp member to render them capable of agglutinating in the presence of the sbp member. For example, if the sbp member to be determined (i.e. "the analyte") is rheumatoid factor (i.e. a heterogeneous population of auto-antibodies binding to the Fc portion of IgG), a suspension containing carbon particles and IgG is prepared. The test result is read by comparing the optical density (measured at a wavelength of 350 to 800 nm) of the assay medium after the test with the optical density of the assay medium before the agglutination test. A change of optical density indicates the presence of the analyte in the sample. In order to avoid self-agglutination of the carbon particles, they are suspended in an aqueous solution of an amino acid, such as glycine, before coating them with the specific binding partner of the sbp member, and the assay is carried out in an assay medium which contains such an amino acid in an amount sufficient to reduce self-aqglutination of the opaque particles.
Also U.S. Pat. No. 5,252,496 (Kang et al.) teaches to subject the carbon particles to a pretreatment with stabilizing agents such as polyalkylene glycol or polysaccharides like dextran to maximize the dispersability of the carbon particles in aqueous media. After this pretreatment with a stabilizing agent, the sbp member is linked to the carbon particle/stabilizing agent complex via a semi-covalently linking reagent such as, e.g., fluorescein-isothiocyanate. The resulting immunochemical label has to be treated subsequently with at least one ionic or non-ionic surfactant in order to render the label suspendable in an aqueous medium such as water or a buffer of low ionic strength.
Bergquist and Waller, J. Immunol. Meth. 61, 33914 344 (1983) disclose a carbon immunoassay (CIA) using carbon particles as contained in India ink to determine the presence, if any, of IgG antibodies in a sample. India ink has specific binding characteristics. It binds, e.g., to rabbit IgG and can be used, therefore, in an assay to detect rabbit IgG antibodies to a particulate antigen. It also binds to the membranes of staphylococci. Said membranes contain protein A which is known to bind human IgG antibodies. These properties can be utilized for a rapid detection of human IgG antibodies to the parasite Toxoplasma gondii. The test comprises mixing active India ink with protein A to prepare a labelled reagent which is then mixed with T. gondii tachyzoites (functioning as the particulate antigen) and a sample suspected of containing human IgG antibodies to T. gondii. The test result may be read under a light microscope. The T. gondii tachyzoites appear black due to adherent carbon particles in the case of a positive CIA reaction, and otherwise remain white. The CIA test is quite insensitive and claimed to be attractive only because of its simplicity.